Introduction
Pinus massoniana , a member of the pinaceae family, is widely distributed in the forest area of central and southern China, and northern Vietnam.1 Due to its fast-growing, strong adaptability and tolerance to barren environments, it plays a critical role in forest ecological integrity restoration and construction.2,3In addition, it is an important economic tree species commonly used for timber, wood pulp, rosin and turpentine, which occupies a vital position in forestry production industry.2,4Besides, some tissues/organs of P. massoniana including needles, bark, pollen and turpentine, and extractives extracted from above parts have been used as traditional Chinese medicines for disease control and treatment purpose.5,6Moreover, P. massoniana is also used in landscape and gardening design, and ornamental horticulture.7
Calcium (Ca) is an essential nutrient element in plants which occurs in plant tissue as free Ca2+ or as Ca2+binding to carboxylic, phosphoric and phenolic hydroxyl groups.8 It has been reviewed that Ca exhibits diverse functions in various plant biological processes and cellular events.9-11 Primarily, it acts as a structure component of plant cell wall and membrane, which impacts cell wall rigidity and cell membrane stability and permeability.9 Thus, plant growth and development progresses can be directly influenced by Ca supply.10 Moreover, it plays an intracellular second messenger role in kinds of plant physiological and biological processes, such as growth and development, ion homeostasis, metabolism regulation, signal transduction and a variety of biotic/abiotic stress response.9-12 The latest studies have demonstrated it also participates in programmed cell death, plant immunity and photosynthesis in plant.13-15 When subjecting to a calcium deficient condition, a variety of disorders and stunned symptoms were observed in horticulture crops.16 However, after applying excessive calcium, plants may suffer Ca toxicity and result in seed germination inhibition and plant growth reduction.16
It was reported that Ca addition alleviated simulated acid rain induced seed germination and seedling growth inhibition in P. massoniana .17 Further proteomic analysis indicated calcium regulated diverse cellular events and biological processes in acid rain treated P. massoniana , and resulted in the tolerance improvement to acid rain.18 Chen et al. demonstrated that exogenous hormones and Ca effectively improved the resistance of P. massoniana to Dendrolimus punctatusattacks.19 Li et al. studied the impact of various Ca concentrations on the growth and physiological index of P. massoniana seedlings, and concluded 1.0-2.0 mM soil Ca concentration is the optimal condition for P. massoniana seedlings growth.20 Nevertheless, the knowledge of molecular regulatory mechanisms of Ca on P. massoniana physiological processes, development and growth is still relatively limited. Considering the important role of P. massoniana in both forestry economy and forest ecosystem, it is of significance to address this issue.
In the present study, the combined physiological and proteomic studies were performed using P. massoniana seedling cultivated at various Ca levels to investigate the impact of exogenous Ca on growth and development, and finally to reveal the underlying molecular mechanisms. In addition, bioinformatics analysis was implemented to demonstrate the biological functions and roles of the differential expressed proteins (DEPs) identified in proteomics experiments, while Ca-related gene expression was analyzed as well. Our study may facilitate the elucidation of the potential regulatory role of Ca in P. massoniana physiology and biology.